Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies. The ciliopathy-linked protein CCDC66 is only known for its ciliary functions. This study reveals that CCDC66 also has extensive non-ciliary functions, localizing to the spindle poles, spindle midzone, central spindle and midbody throughout cell division, where it regulates mitosis and cytokinesis by promoting microtubule nucleation and organization. Faithful segregation of genetic and cytoplasmic material during cell division is essential for growth and development of multicellular organisms. Deregulation of the molecular processes that regulate cell division leads to aneuploidy and chromosomal instability and thereby to the initiation and progression of various human cancers ,2. As suIn animal somatic cells, MT nucleation is initiated at the centrosomes, the preexisting spindle MTs, and the chromatin, with centrosomes being the major MT-organizing centers ,11. The Organization of MTs into highly ordered mitotic and cytokinetic arrays play critical roles for cell division. For example, 20 to 40 kinetochore MTs in human cells form parallel bundles termed K-fibers, which run from spindle poles to kinetochores and are essential for chromosome alignment and segregation –29. BridThe drastic remodeling of the MT network during cell division requires precise regulation of when and where MAPs are activated. A subset of MAPs is active during mitosis but inactive during interphase. Such regulation is achieved by modulation of their affinity to MTs, regulation of their cellular abundance, and localization and posttranslational modifications . Importa−/− mouse . CC. CC59]. The localization of CCDC66 to spindle microtubules suggests that mitotic progression defects associated with its depletion might be a consequence of defective spindle assembly and orientation. To investigate this, we analyzed various spindle properties in control and CCDC66-depleted cells. First, we measured the angle between the spindle axis and the substratum, which revealed an increase from 7.7 ± 0.3° in control cells to 13.1 ± 1.5° in CCDC66-depleted cells . By takiProper formation and organization of K-fibers is essential for chromosome alignment and segregation, suggesting that chromosome-related defects in CCDC66-depleted cells could be due to impaired K-fibers ,63. To dCCDC66 localizes to the central spindle, intercellular bridge/midbody, and K-fibers, which are highly organized and stable MT bundles. Moreover, it interacts and co-localizes with PRC1, a well-characterized regulator of MT bundling during cell division . These lQuantification of the MT fluorescence intensity along the pole-to-pole axis in anaphase cells showed that MT intensity was reduced at the central spindle in CCDC66-depleted cells . This reDefects in central spindle and intercellular bridge/midbody, as well as K-fibers could be attributed to MT bundling activity of CCDC66. To test this, we used in vitro experiments to investigate the possible MT cross-linking activity of CCDC66. We expressed MBP-mNG-CCDC66 in insect cells and purified it using nickel beads . As a coMT-binding and bundling activities of PRC1 are required for microtubule organization at the spindle midzone in anaphase, localization of MAPs within this structure and successful completion of cytokinesis ,67–69. GOne of the most prominent phenotypes associated with CCDC66 depletion is the significant decrease in astral, metaphase spindle, and central spindle MT intensities. This finding led us to investigate the functions of CCDC66 during MT nucleation in dividing cells. To this end, we performed MT regrowth experiments in control and CCDC66-depleted cells synchronized using the Eg5-inhibitor S-trityl-L-cysteine (STLC) . FollowiDuring centrosome maturation, centrosomes recruit more PCM proteins to increase their MT-nucleating capacity. CDK5RAP2, CEP192, CEP152, and pericentrin are required for centrosomal recruitment of gamma-tubulin ,71. GiveThe functional significance of the dynamic localization of CCDC66 to the centrosomes and various MT arrays as well as the relative contribution of its MT nucleation and organization activities to its functions are not known. To distinguish between the function of these different CCDC66 pools and activities during cell division, we performed phenotypic rescue experiments with 3 different CCDC66 siRNA-resistant constructs: (1) mNG-CCDC66 to validate the specificity of the phenotypes; (2) mNG-CCDC66 (570–948) to assess the functional significance of CCDC66 localization to the centrosomes and MT-binding and bundling activity; and (3) C-terminal mNG-CCDC66 fusion with the centrosomal targeting domain PACT to distinguish its centrosome-specific activities from the ones mediated by MTs. Next, we used lentiviral transduction to generate U2OS cells stably expressing these fusion proteins as well as mNG itself as a control and validated their expression at the expected size, siRNA resistance, and localization in CCDC66-depleted cells by immunoblotting and immunofluorescence Figs , and S7BNext, we examined whether expression of mNG-fusions of CCDC66, CCDC66 (570–948), and CCDC66-PACT restores defective targeting of gamma-tubulin to the centrosomes, reduced spindle MT intensity in metaphase and anaphase cells, increased cold-sensitivity of K-fibers, spindle misorientation, disorganized central spindle, and increased binucleation in CCDC66-depleted cells. mNG-CCDC66 expression rescued all 7 phenotypes to comparable or greater levels to control siRNA-transfected mNG-expressing cells, indicating that these phenotypes are specific to CCDC66 depletion Figs . SimilarIn this study, we identified the centrosomal and ciliary MAP, CCDC66, as a new player of the machinery governing the assembly and organization of the mitotic and cytokinetic MT arrays and thereby cell cycle progression. As summarized in the model shown in The results of our study revealed MT nucleation and organization as the 2 major mechanisms by which CCDC66 functions during cell division. In mitotic cells, CCDC66 regulates centrosome maturation via recruitment of core PCM proteins and is required for MT nucleation from the centrosomes during bipolar spindle assembly and orientation. The inability of centrosome-restricted mNG-CCDC66-PACT to rescue central spindle, cytokinesis, and K-fiber defects show that CCDC66 does not regulate these processes via centrosomal MT nucleation. Furthermore, 2 lines of evidence indicate that CCDC66 is also involved in MT-mediated MT nucleation during spindle assembly. First, depletion of CCDC66 resulted in a decrease in microtubule nucleation from noncentrosomal nucleation centers in mitotic cells as well as in gamma-tubulin recruitment to the spindle. Second, CCDC66 depletion resulted in decreased MT density of central spindle in anaphase, which represents de novo noncentrosomal MT generation in inter-chromosomal region and requires MT-associated proteins that participate in nucleation, stabilization, and bundling ,73. The The second mechanism by which CCDC66 operates during mitosis is organization of MTs via its cross-linking activity. Using in vitro and cellular experiments, we showed that CCDC66 bundles MTs in part via its C-terminal MT-binding domain. In line with our results, a recent study published during the revision of our manuscript reported that GFP-CCDC66 purified from mammalian cells binds to MTs and bundles them in vitro . BundlinDespite its roles in MT nucleation and organization, CCDC66 depletion did not result in shorter spindles, which can be explained by a compensatory mechanism activated in CCDC66-depleted cells. Spatiotemporal regulation of MT polymerization, depolymerization, and sliding is critical to spindle length maintenance, providing remarkable ability of metaphase spindles to correct transient fluctuations in morphology ,75. OtheThe pleiotropic localization and activities of CCDC66 during cell division present challenges in specifically defining mechanisms that underlie its mitotic and cytokinetic functions. Are they regulated by centrosomal and microtubule-associated pools of CCDC66 or their coordinated activity? While we first aimed to address this via identification of CCDC66 fragments that contribute to a single localization and activity, we could not identify such regions. As an alternative, we performed phenotypic rescue experiments with CCDC66 (570–948), which bundles MTs and localizes to centrosomes and CCDC66-PACT, which exclusively localizes to the centrosome. Of note, the strong centrosome affinity of the PACT domain increased CCDC66 levels at the centrosome and created more binding sites for its interactors such as gamma-tubulin, which might have compensated for lack of MT association for a subset of CCDC66 functions. Defective spindle MT density, recruitment of gamma-tubulin to the centrosomes, and spindle orientation were rescued both by CCDC66-PACT and CCDC66 (570–948), albeit to different extents. These results suggest that centrosomal and MT pools cooperate in these processes. Strikingly, K-fiber, central spindle, and cytokinesis defects were rescued only by CCDC66 (570–948). Given the essential role of chromosome and MT-dependent MT nucleation and bundled MT arrays of the central spindle and midbody during cytokinesis, these results suggest that CCDC66-mediated MT bundling and noncentrosomal MT nucleation are required for these cellular processes ,77.−/− mice were embryonically viable and did not develop tumors, indicating that defective cell cycle defects linked to CCDC66 loss alone do not disrupt embryogenesis and is not tumorigenic [CCDC66 has been implicated in several developmental disorders including retinal degeneration and Joubert syndrome –53. Consorigenic .This migRR plasmids used for transfection and transformation experiments were previously described [RR plasmid and cloned into pCDH-EF1-mNeonGreen-T2A-Puro plasmid. EB3-mNeonGreen-T2A-gamma-tubulin-tagRFP plasmid was a gift from Andrew Holland (Johns Hopkins University School of Medicine) [pDEST-GFP-CCDC66 and pDEST-GFP-CCDC66escribed . Full-leedicine) . Myc-Biredicine) . PLK1 waHuman telomerase immortalized retinal pigment epithelium cells  were cultured with Dulbecco’s modified Eagle’s Medium DMEM/F12 50/50 medium (Pan Biotech) supplemented with 10% fetal bovine serum  and1% penicillin-streptomycin (Gibco). Human embryonic kidney  and osteosarcoma epithelial  cells were cultured with DMEM medium (Pan Biotech) supplemented with10% FBS and 1% penicillin-streptomycin. All cell lines were authenticated by Multiplex Cell Line Authentication (MCA) and were tested for mycoplasma by MycoAlert Mycoplasma Detection Kit (Lonza). U2OS cells were transfected with the plasmids using Lipofectamine 2000 and according to the manufacturer’s instructions (Thermo Scientific). HEK293T cells were transfected with the plasmids using 1 mg/ml polyethylenimine, MW 25 kDa (PEI). For microtubule depolymerization experiments, cells were treated with 5 μg/ml nocodazole (Sigma-Aldrich) or vehicle (dimethyl sulfoxide) for 1 h at 37°C. For cell synchronization experiments, 5 μm (+)-S-trityl-L-cysteine (STLC) (Alfa-Aesar) was used for 16 h at 37°C.5 U2OS cells were seeded on 6-well tissue culture plates the day before infection, which were infected with 1 ml of viral supernatant the following day. Approximately 48 h post-infection, cells were split and selected in the presence of 6 mg/ml puromycin for RPE1s and 4 mg/ml puromycin for U2OS cells for 4 to 6 days until all the control cells died. U2OS and RPE1 cells stably expressing mCherry-H2B and mNeonGreen fusions of full-length CCDC66, and its truncations were generated by infection of cells with lentivirus expressing the fusions.Lentivirus were generated using pcDH-mNG-CCDC66, pcDH-mNG-CCDC66 (570–948), pcDH-mNG-CCDC66-PACT, and pCDH-EF1-mNeonGreen-T2A-Puro, and pLVPT2-mCherry-H2B plasmids as transfer vectors. For infection, 1 × 10CCDC66 was depleted using a siRNA with the sequence 5′-CAGTGTAATCAGTTCACAAtt-3′. Silencer Select Negative Control No. 1 (Thermo Scientific) was used as a control . siRNAs 6 cell/ml) were infected with P1 baculovirus produced in Sf9 cells, carrying His-MBP-mNeonGreen-CCDC66, at MOI of 1. Cells were collected 48 h after infection.Protein expression in Rosetta (BL21(DE3)) cells transformed with respective constructs was induced by addition of 0.5 mM IPTG at OD600 of 0.5 to 0.6 for 16 h at 18°C. BEVS baculovirus expression system and protocol was used for expression of tagged full-length CCDC66 protein. Briefly, 100 ml of Hi5 cells (1 × 102).For protein purification, cells were lysed by sonication in lysis buffer (20 mM Hepes pH 7.0 (or pH 7.5 for full-length protein), 250 mM NaCl, 0.1% Tween20, 2 mg/ml lysozyme, 1 mM PMSF, 1 mM protease inhibitor cocktail, 5 mM BME, and 10 mM Imidazole) and clarified at 19,000 rpm for 1 h at 4°C. His-tagged proteins were subsequently purified using Ni-NTA–agarose beads (Thermo Scientific). Proteins were eluted with elution buffer (20 mM Hepes (pH 7.0 or pH 7.5), 250 mM NaCl, 5 mM BME, and 250 mM imidazole). For subsequent microtubule assays, proteins were dialyzed against BRB80 buffer (80 mM PIPES (pH 6.8), 1 mM EGTA, 1 m M MgClFluorescent MTs were polymerized at 2 mg/ml by incubating tubulin and rhodamine-labeled tubulin (Cytoskeleton) at 10:1 ratio in BRB80 with 1 mM DTT and 1 mM GTP for 5 min on ice, then preclearing by centrifuging for 10 min at 90,000 rpm at 2°C in TLA100 rotor. Cleared tubulin mixture was polymerized at 37°C by adding taxol and increasing concentration stepwise, with final concentration of 20 μm. MTs were pelleted over warm 40% glycerol BRB80 cushion at 70,000 rpm for 20 min. After washes with 0.5% Triton-X100, pellet was resuspended in 80% of the starting volume of warm BRB80 buffer with 1 mM DTT and 20 μm taxol.2, 1 mM DTT, protease inhibitors) for 30 min rocking at room temperature. The reaction mixtures were transferred into a flow chamber under an HCl-treated coverslip, and unstuck proteins were washed out with the excess of buffer T. Bundling of fluorescent MTs was observed with a Leica SP8 confocal microscope and 63× 1.4 NA oil objective (Leica Mycrosystems). Experiment was repeated 4 times.Bundling assays were performed as previously described . BrieflyPurified bovine brain tubulin (Cytoskeleton) was precleared at 90,000 rpm for 5 min at 2°C with TLA100 rotor. Cleared tubulin was polymerized at 1 mg/ml in the presence of 1 mM GTP and increasing concentrations of taxol to a final concentration of 20 μm. After incubation of microtubules at room temperature overnight, 500 nM of purified protein was mixed with 3 μm taxol-stabilized microtubules or BRB80 buffer with taxol and incubated for 30 min at room temperature. Samples were loaded onto 60% glycerol BRB80 cushions and centrifuged at 50,000 rpm for 30 min with TLA100 rotor at room temperature. Supernatants were collected, glycerol cushions removed by aspiration, and pellets solubilized in SDS sample buffer. Equivalent volumes of supernatant and pellet fractions were resolved by SDS-PAGE.U-ExM was performed as previously described . BrieflyCells were grown on coverslips, washed twice with PBS, and fixed in either ice cold methanol at −20°C for 10 min or 4% PFA in Cytoskeletal Buffer ((100 mM NaCl (Sigma-Aldrich), 300 mM sucrose (Sigma-Aldrich), 3 mM MgCl2 (Sigma-Aldrich), and 10 mM PIPES (Sigma-Aldrich)). For CCDC66 endogenous staining with the rabbit polyclonal antibody, cells were first fixed with methanol at −20°C, then with 100% acetone for 1 min at room temperature. After rehydration in PBS, cells were blocked with 3% BSA (Capricorn Scientific) in PBS followed by incubation with primary antibodies in blocking solution for 1 h at room temperature. Cells were washed 3 times with PBS and incubated with secondary antibodies and DAPI (Thermo Scientific) at 1:2,000 for 45 min at room temperature. Following 3 washes with PBS, cells were mounted using Mowiol mounting medium containing N-propyl gallate (Sigma-Aldrich). Primary antibodies used for immunofluorescence were rabbit anti-CCDC66 , mouse anti gamma-tubulin  at 1:1,000, rabbit anti GFP at 1:2,000 (custom made) , mouse aTime lapse live imaging was performed with Leica SP8 confocal microscope equipped with an incubation chamber. For cell cycle experiments, asynchronous cells were imaged at 37°C with 5% CO2 with a frequency of 6 min per frame with 1.5-mm step size and 12-mm stack size in 512 × 512 pixel format at a specific position using HC PL FLUOTAR 20×/0.50 DRY objective. For centrosomal protein level quantifications, images were acquired with Leica DMi8 inverted fluorescent microscope with a stack size of 8 mm and step size of 0.3 mm in 1,024 × 1,024 format using HC PL APO CS2 63× 1.4 NA oil objective. Higher resolution images were taken by using HC PL APO CS2 63× 1.4 NA oil objective with Leica SP8 confocal microscope.2 area centered on the centrosome in each cell. Total pixel intensity of fluorescence within the region of interest was measured using ImageJ . Background subtraction was performed by quantifying fluorescence intensity of a region of equal dimensions in the area proximate to the centrosome. Statistical analysis was done by normalizing these values to their mean.Quantitative immunofluorescence for CEP192, CEP152, CDK5RAP2, pericentrin, gamma-Tubulin, PRC1, and alpha-tubulin was performed by acquiring a z stack of control and depleted cells using identical gain and exposure settings. The centrosome region for each cell was defined by staining for a centrosomal marker including gamma-tubulin. The region of interest that encompassed the centrosome was defined as a circle 3.4-mm−1(h/L)/π where h represents the z stack difference between 2 centrosomes, L represents the distance between centrosomes. Spindle length is calculated by the formula SL = √(h2+L2), where h represents the stack difference between 2 centrosomes, L represents the distance between centrosomes. Centrosome width at metaphase is calculated by measuring the length of the PCM of the centrosomes as described in the illustrations in U2OS cells were grown on coverslips and transfected with control and CCDC66 siRNA, fixed with methanol, and stained for gamma-tubulin. Images are acquired with Leica DMi8 inverted fluorescence microscope at 1,024 × 1,024 format with 0.3-mm z step size. Spindle angle is calculated by the formula α = 180*tanU2OS cells were grown on coverslips and transfected with control and CCDC66 siRNA, fixed with methanol, and stained for alpha-tubulin. Images were acquired with Leica SP8 Confocal microscopy at 1,024 × 1,024 format with 2× zoom factor. For quantification of the astral microtubule intensity, 5 ROIs having XY size 2 microns were positioned manually on the astral microtubules and intensity was recorded. Background of the same ROI was measured in cytoplasm and subtracted from the average signal intensity. For astral microtubule length, the longest microtubule was measured using the length tool on ImageJ. For quantification of spindle microtubule intensity, 10 ROIs having XY size 2 microns were positioned manually on the spindle microtubules and intensity was recorded. Background of the same ROI was measured in cytoplasm and subtracted from the average signal intensity. The values of control and CCDC66 siRNA-treated metaphase cells were plotted relative to mean intensity of control siRNA. For K-fiber stability assay, control and CCDC66-depleted cells were placed on ice (4°C) for 10 min without previous treatment and cell synchronization with proteasome inhibitor MG132. Cells were washed extensively with cold PBS to prevent MT polymerization, and then fixed with ice cold methanol at −20°C for 3 min. The tubulin fluorescence intensity of cold stable K-fibers was measured as described above for quantification of spindle microtubule intensity.U2OS cells were grown on coverslips and transfected with control and CCDC66 siRNA, fixed with methanol, and stained for alpha-tubulin. Images were acquired with Leica SP8 Confocal microscopy at 1,024 × 1,024 format with 2× zoom factor. For quantification of central spindle microtubule intensity, 10 ROIs having XY size 2 microns were positioned manually on the central spindle microtubules and intensity was recorded. Background of the same ROI was measured in cytoplasm and subtracted from the average signal intensity. Aberrant central spindles are judged from the disorganization of the microtubules.U2OS cells were grown on poly-L-lysine coated coverslips and treated with control or CCDC66 siRNA. After 48 h of transfection, cells were treated with 5 μm/ml STLC for 16 h. Next day, cells were treated with 5 μg/ml nocodazole for 1 at 37°C. Cells were washed extensively with cold PBS to prevent MT polymerization then incubated with warm media and fixed at indicated time points and stained for alpha-tubulin and centrin. Images were acquired with Leica SP8 Confocal microscopy at 1,024 × 1,024 format with 2× zoom factor. To quantify MT nucleation area (aster size), polygon selection tool on ImageJ is used. Centrosomal and noncentrosomal MT nucleation points were defined based on centrin staining, which marks the centrioles.HEK293T cells were co-transfected with indicated plasmids. Approximately 48 post-transfection, cells were washed and lysed with lysis buffer (50 mM HEPES (pH 8), 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, Protease inhibitors 1:100 pro-block PIC + 1:100 PMSF) for 45 min. Lysates were centrifuged at 13,000 rpm for 10 min at 4°C, and supernatants were transferred to a tube. A total of 100 μl from each sample was saved as input. The rest of the supernatant was immunoprecipitated with anti-FLAG M2 agarose beads (Sigma-Aldrich) overnight at 4°C. After washing 3× with lysis buffer, samples were resuspended in SDS containing sample buffer and analyzed by immunoblotting.g for 15 min. The protein concentration of the resulting supernatants was determined with the Bradford solution . For immunoblotting, equal quantities of cell extracts were resolved on SDS-PAGE gels, transferred onto nitrocellulose membranes, blocked with TBST in 5% milk for 1 h at room temperature. Blots were incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4°C, washed with TBST 3 times for 5 min and blotted with secondary antibodies for 1 h at room temperature. After washing blots with TBST 3 times for 5 min, they were visualized with the LI-COR Odyssey Infrared Imaging System and software at 169 mm (LI-COR Biosciences). Primary antibodies used for immunoblotting were mouse anti gamma-tubulin  at 1:5,000, rabbit anti GFP at 1:10,000 (homemade), mouse anti alpha-tubulin  at 1:5,000, rabbit anti CEP152  at 1:1,000, rabbit anti-CEP192  at 1:1,000, rabbit anti-Cdk5Rap2  at 1:1,000, rabbit anti-pericentrin  at 1:2,000, mouse anti-mNG  and mouse anti-CCDC66 (sigma SAB1408484) at 1:500. Secondary antibodies used for western blotting experiments were IRDye680- and IRDye800-coupled and were used at 1:15,000 (LI-COR Biosciences). Secondary antibodies used for western blotting experiments were IRDye680- and IRDye800-coupled and were used at 1:15,000 (LI-COR Biosciences)Cells were lysed in 50 mM Tris (pH 7.6), 150 mM NaCI, 1% Triton X-100, and protease inhibitors for 30 min at 4°C followed by centrifugation at 15.000t tests and 1-way analysis of variance (ANOVA) were applied to compare the statistical significance of the measurements. For data that does not follow normal distribution, we applied nonparametric Mann–Whitney test. Error bars reflect SD. Following key is followed for asterisk place holders for p-values in the figures: *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001, ns: not significant.Data were analyzed and plotted using GrapPad Prism 7 . Results are shown as mean ± standard errors (SEM) of the mean. Numbers of biological replicates are indicated in the figure legends. Two-tailed unpaired S1 Fig(A) Localization of CCDC66 at different stages of the cell cycle. U2OS were fixed with methanol followed by acetone and stained for CCDC66, alpha-tubulin, and DAPI. Scale bar: 5 μm, insets show 4× magnifications of the boxed regions. (B) Validation of RPE1::mNeonGreen-CCDC66 expression with antibody. RPE1::mNeonGreen-CCDC66 stable cell line was fixed with methanol and stained with CCDC66 antibody and alpha-tubulin. Insets show 4× zoom. Scale bar: 5 μm. (C) Validation of mNeonGreen-CCDC66 expression in U2OS::mNeonGreen-CCDC66 and RPE1::mNeonGreen-CCDC66 stable lines by immunoblotting. Extracts from cells were prepared, resolved by SDS-PAGE and blotted with mNeonGreen antibody. (D) Relative expression level of mNeonGreen-CCDC66 compared to endogenous protein in U2OS cells. Extracts from cells were prepared, resolved by SDS-PAGE and blotted with CCDC66 antibody. (E) Localization of mNeonGreen-CCDC66 at different stages of cell cycle. RPE1 cells stably expressing mNeonGreen-CCDC66 fusion (RPE1::mNeonGreen-CCDC66) were fixed with 4% PFA and stained for alpha-tubulin and DAPI. Scale bar: 5 μm. (F) Dynamic localization of mNeonGreen-CCDC66 throughout the cell cycle. U2OS cells stably expressing mNeonGreen-CCDC66 fusion (U2OS::mNG-CCDC66) were incubated with 100 nM SiR-Tubulin overnight. Images are acquired every 4 min using confocal microscopy. Shown are sixteen time-lapse images from (TIF)Click here for additional data file.S2 Figp-value (Fisher’s exact test) of GO terms. The data underlying the graphs showing the plot profiles in the figure can be found in (A) Localization of mNeonGreen-CCDC66 in RPE1 cells relative to PRC1 during anaphase. RPE1::mNeonGreen-CCDC66 cells were fixed with 4% PFA and stained for PRC1 and DAPI. Graphs show the plot profiles to assess co-localization with the indicated marker. Using ImageJ, a straight line was drawn on the midbody, and intensity along the distance was plotted on Graphpad Prism.  GO-enrichment analysis of the CCDC66 proximity interactors based on their (B) biological process and (C) cellular compartment. The x-axis represents the log-transformed (TIF)Click here for additional data file.S3 Fig(A) Validation of the efficiency of RNAi-mediated CCDC66 depletion by western blotting and immunofluorescence. U2OS cells were transfected with siControl or siCCDC66. Approximately 48 h after post-transfection, cells were fixed and stained with the indicated antibodies. In parallel, cell extracts immunoblotted for CCDC66 and vinculin (loading control). Band intensities were measured on ImageJ and normalized against background and vinculin intensities. Arbitrary value is determined based on siControl. (B) Validation of the CCDC66 antibody by immunofluorescence. U2OS cells were transfected with control or CCDC66 siRNA, fixed with methanol followed by acetone 48 h post-transfection and stained for CCDC66 and alpha-tubulin. Representative images are shown at different stages of the cell cycle to indicate the decrease in the signal of CCDC66 upon siCCDC66 transfection. Insets show 4× zoom of boxed areas. Scale bar: 5 μm. (C) Quantification of (TIF)Click here for additional data file.S4 Fig(A) Cellular abundance of tubulin in control and CCDC66-depleted cells. Cells are transfected with either control or CCDC66 siRNA. After 48 h, the lysates are collected and immunoblotted for alpha-tubulin, CCDC66, and vinculin (loading control). (B) Quantification of (A). Data represent mean ± SEM of 2 independent experiments. (ns: not significant). (C) CCDC66 localizes on K-fibers. U2OS cells were grown on coverslips and incubated in ice for 10 min before fixation with methanol followed by acetone. Cells were stained for CCDC66 and ACA. Inset shows 4× zoom of boxed area. Scale bar: 5 μm. The data underlying the graphs shown in the figure can be found in (TIF)Click here for additional data file.S5 Fig(A) Spindle midzone length is not altered by CCDC66 depletion. U2OS cells were transfected with control or CCDC66 siRNA, fixed with methanol followed 48 h post-transfection and stained for alpha-tubulin and DAPI. As shown in the representation, midzone length was measured as the distance between the most distant microtubule ends in the midzone. Data represent the mean ± SEM of 4 independent experiments. (ns: not significant). (B) CCDC66 depletion does not alter PRC1 midbody levels. U2OS cells were transfected with siRNA then fixed with methanol after 48 h and stained for PRC1, alpha-tubulin, and DAPI. Images represent cells from anaphase and cytokinesis. Scale bar: 5 μm. For quantification, PRC1 intensity was measured on ImageJ, the background signal was subtracted, and normalized value was multiplied with area. Arbitrary value was determined by normalizing against siControl. (ns: not significant). (C) His-MBP-mNeonGreen-CCDC66 purification. His-MBP-mNeonGreen-CCDC66 was purified from insect cells using Ni-NTA agarose beads. Coomassie staining shows the proteins in pellet, initial sample, flowthrough, wash, and elutions. (D) Validation of His-MBP-mNeonGreen-CCDC66 purification. Purified His-MBP-mNeonGreen-CCDC66 purification was run on SDS-PAGE and blotted with CCDC66 antibody. Arrow corresponds to the full-length His-MBP-mNeonGreen-CCDC66. (E) His-MBP-mNeonGreen-CCDC66 directly interacts with microtubules. His-MBP-mNeonGreen-CCDC66 was purified from insect cells, and in vitro microtubule pelleting was performed and visualized by Coomassie staining. BSA was used as negative control. S stands for supernatant, P stands for pellet. (F) Validation of MBP-mNeonGreen purification with Coomassie. (G) Validation of MBP-CCDC66 (570–948) purification with Coomassie. MBP-His-CCDC66 (570–948) was purified from bacterial culture using Ni-NTA agarose beads. Purified protein was run on SDS-PAGE. Coomassie staining and western blotting with anti-His antibody shows the purified protein. (H) MBP-CCDC66 (570–948) directly interacts with microtubules. MBP-His-CCDC66 (570–948) was purified from bacterial culture using Ni-NTA agarose beads and microtubule pelleting was performed. Coomassie staining is presented. S stands for supernatant, P stands for pellet. (I) Effect of mNeonGreen-CCDC66 and mNeonGreen-CCDC66 (570–948) overexpression on microtubule organization and stability in cells. U2OS cells were transfected with mNeonGreen, GFP-CCDC66, or mNG-CCDC66 (570–948) expression plasmids. Approximately 24 h post-transfection, cells were treated with 5 μm nocodazole or 0.01% DMSO for 1 h, fixed with methanol, and stained for the indicated proteins and DNA. Scale bar: 5 μm. The data underlying the graphs shown in the figure can be found in (TIF)Click here for additional data file.S6 Figp < 0.01) Scale bar: 5 μm. (B) Effect of CCDC66 depletion on the cellular abundance of PCM proteins. U2OS cells were transfected with siControl or siCCDC66, and 48 h after transfection extracts from cells were immunoblotted for CDK5RAP2, CEP192, CEP152, gamma-tubulin, pericentrin and vinculin (loading control), or GAPDH (loading control). Band intensities were measured on ImageJ and normalized against background and vinculin intensities. Data represent the mean ± SEM of 3 independent experiments. (C) U-ExM analysis of control and CCDC66 depleted cells. U2OS cells were transfected with control and CCDC66 siRNA. Approximately 48 h post-transfection, cells were synchronized by 16 h STLC treatment and prepared for imaging. Cells were stained for gamma-tubulin and acetylated tubulin, imaged using confocal microscopy and deconvolved. Mitotic and G2 cells were picked for representation.  Effects of CCDC66 depletion on abundance of PCM proteins at the centrosomes. U2OS cells were transfected with control and CCDC66 siRNA. After 48 h, cells were fixed with methanol and stained for (D) CEP192 and (E) CEP152. Centrosomal abundance of PCM proteins was measured as described in (A) Quantification of microtubule nucleation sites following nocodazole washout of STLC-synchronized control and CCDC66-depleted cells. Representative images are shown for control and CCDC66-depleted cells. The graph indicates the number of microtubule nucleation points that are counted from Centrin 3 and alpha-tubulin signals. Data represent the mean ± SEM of 2 independent experiments. (**(TIF)Click here for additional data file.S7 Fig−1(h/L)/π where h represents the stack difference between 2 centrosomes, L represents the distance between centrosomes when projected onto 1 z plane. Data represent the mean ± SEM of 2 independent experiments. (**p < 0.01 ***p < 0.001 ****p < 0.0001). (D) Representative images for the K-fiber rescue experiment performed using U2OS::mNeonGreen, U2OS::mNeonGreen-CCDC661-948, U2OS::mNeonGreen-CCDC66-PACT, and U2OS::mNeonGreen-CCDC66570-948 stable cells. Cells were transfected with control and CCDC66 siRNA. Approximately 48 h post-transfection, they were fixed with methanol and stained for alpha-tubulin and DAPI. Scale bar: 5 μm. (E) Quantification of p < 0.0001, ns: not significant). (F) Quantification of p < 0.0001, ns: not significant). The data underlying the graphs shown in the figure can be found in (A) Validation of U2OS cells lines that stably express siRNA-resistant mNeonGreen-CCDC66, mNeonGreen-CCDC66-PACT, and mNeonGreen-CCDC66 (570–948). Extracts from cells were prepared, resolved by SDS-PAGE, and blotted with mNeonGreen antibody. (B) Validation of siRNA resistance of the CCDC66 rescue constructs. U2OS cells were transfected with control and CCDC66 siRNA. Approximately 48 h post-transfection extracts from cells were prepared, resolved by SDS-PAGE, and blotted with CCDC66 antibody. The red arrowheads indicate endogenous CCDC66, which is masked due to higher expression of the fusion proteins and high background associated with the CCDC66 antibody. The green arrowheads indicate the mNeonGreen fusions of CCDC66. (C) Quantification of (TIF)Click here for additional data file.S1 Raw ImagesRed boxes are used to show the represented blot in the figures. Red crosses show the parts of the blot that are not used in the figures.(PDF)Click here for additional data file.S1 DataEach tab in the file represents the corresponding figure panel produced based on the displayed data.(XLSX)Click here for additional data file.S1 MovieImages are taken every 2 min. Scale bar: 5 μm.(AVI)Click here for additional data file.S2 MovieImages are taken every 4 min. Scale bar: 5 μm.(AVI)Click here for additional data file.S3 MovieImages were taken every 6 min for 16 h. Scale bar: 5 μm.(AVI)Click here for additional data file.S4 MovieImages were taken every 6 min for 16 h. The movie represents a CCDC66 depleted cell going through normal mitotic progression. Scale bar: 5 μm.(AVI)Click here for additional data file.S5 MovieImages were taken every 6 min for 16 h. The cell marked with an asterisk represents a CCDC66 depleted cell going through apoptosis after delayed mitosis. Scale bar: 5 μm.(AVI)Click here for additional data file.S6 MovieImages were taken every 6 min for 16 h. This movie represents a CCDC66 depleted cell going through premature mitotic exit. Scale bar: 5 μm.(AVI)Click here for additional data file.S7 MovieImages were taken every 6 min for 16 h. This movie represents a CCDC66 depleted cell with prometaphase arrest and nonpersistent metaphase plate. Scale bar: 5 μm.(AVI)Click here for additional data file.S8 MovieImages were taken every 6 min for 16 h. This movie represents a CCDC66 depleted cell with a cytokinesis defect. Scale bar: 5 μm.(AVI)Click here for additional data file.S9 MovieImages were taken every 6 min for 16 h. This movie represents a CCDC66 depleted cell with a cytokinesis defect. Scale bar: 5 μm.(AVI)Click here for additional data file. 29 Oct 2021Dear Dr Firat-Karalar, Thank you for submitting your manuscript entitled "CCDC66 controls mitotic progression and cytokinesis by promoting centrosome maturation and microtubule bundling" for consideration as a Research Article by PLOS Biology. Please accept my apologies for the delay in getting back to you as we consulted with an academic editor about your submission.Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed the checks it will be sent out for review. 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We apologise in advance for any inconvenience caused and will do our best to minimize impact as far as possible.plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,RichardRichard Hodge, PhDAssociate Editor, PLOS Biologyrhodge@plos.orgPLOSEmpowering researchers to transform scienceCarlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdomplosbio.org I @PLOSBiology I BlogORCiD I California (U.S.) corporation #C2354500, based in San Francisco 4 Nov 2021AttachmentPointByPointResponse_final.pdfSubmitted filename: Click here for additional data file. 16 Dec 2021Dear Dr Firat-Karalar,Thank you very much for submitting your manuscript "The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting centrosome maturation and microtubule bundling" for consideration as a Research Article at PLOS Biology. Please accept my sincere apologies for the delays that you have experienced during the peer review process. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by four independent reviewers.The reviews are attached below. You will see that the two previous reviewers from the Life Science Alliance note that the addition of the endogenous CCDC66 data has improved the manuscript, but ask that the previously suggested experiments are addressed according to the revision plan provided in the rebuttal. After discussions with the Academic Editor, we agree with Reviewer #3 that the proposed phenotypic rescue experiments are essential to consider a revision of the manuscript.We also recruited two additional reviewers, as we previously mentioned. While they find the work potentially interesting, they raise concerns regarding several statements over-interpretating the results. They also think that additional mechanistic data is needed to directly assign a role for CCD66 during cytokinesis and microtubule nucleation.In light of the reviews, we will not be able to accept the current version of the manuscript. We remain interested in your study and we would be willing to consider resubmission of a comprehensively revised version that thoroughly addresses all the reviewers' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would be sent for further evaluation by the reviewers.We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you six months to revise your manuscript. We expect to receive your revised manuscript within 6 months.plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. 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For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5Please note that as a condition of publication PLOS' data policy  such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,RichardRichard Hodge, PhDAssociate Editor, PLOS Biologyrhodge@plos.orgPLOSEmpowering researchers to transform scienceCarlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdomplosbio.org I @PLOSBiology I BlogORCiD I California (U.S.) corporation #C2354500, based in San Francisco*****************************************************REVIEWS:Reviewer #1: In this manuscript, the authors examine the contribution of the ciliopathy protein CCDC66 to mitosis and cytokinesis. The authors show that the protein localizes to mitotic microtubules in dividing cells including spindle kinetochore fibers, PCM and midbody using both immunofluorescence and expression of the tagged exogenous protein. CCDC66 contribution to the localization of other PCM components, binding partners and microtubule crosslinking activity of CCD66 are determined. For the most part the work provides new information that will be of interest to cell biologists. 1. To study CCD66 in mitotic cells, the authors deplete the protein with siRNA, which is verified with an immunoblot and by immunofluorescence. To more clearly depict the mitotic fate of the cells that were depleted of CCDC66, it would be good to show each cell as a bar representing the duration of mitosis and indicating the fate. An example of this type of representation see Wong, et al Science 2015, Figure 4. 2. The defects in astral microtubules and spindle positioning and the localization of CCDC66 at the centrosome are consistent with a role for CCDC66 in microtubule nucleation. This is not a surprising result for a protein at the PCM that binds to gamma tubulin. The images of astral microtubules in the stained cells are not particularly convincing, and better images would be nice to see. 3. Given the ability of CCDC66 to bind and bundle microtubules and its co-localization with PRC1 in the midbody, the authors hypothesize that CCDC66 contributes to cytokinesis. Consistent with this, the authors state that a large percentage of cells depleted for CCDC66 display asymmetric furrows indicating a defect in cytokinesis. However, I had some questions about other aspects of cytokinesis. Midzone localization in anaphase cells is weak - it is present in the expressing cells but not apparent in the stained cells. This should be noted or clarified. CCDC66 interacts with PRC and co-localizes with PRC at the midbody in late telophase/cytokinesis. Do CCDC66 and PRC1 co-localize at the anaphase midzone? Given the staining in Fig 1, I think not. Please clarify. The additional "spots" of fluorescence of CCDC66 in the midzone  should be quantified to be sure these are not aggregates of the expressed protein. What % of cells show these? Are they observed in fixed and stained cells or only the expressing cells? The data in Fig S3 show midzone microtubules, in control and CCDC66 depleted cells, but defects but not quantified. The examples suggest subtle differences given the cell-to-cell variation in midzone microtubule organization. This needs to be quantified or removed. The cytokinesis defects could result from defects in spindle formation that are manifest later on at cytokinesis. Another possibility is that CCDC66 contributes not to midzone organization in anaphase, but in the abscission process.4. The authors use microtubule regrowth assays to ask if the loss of CCDC66 impacts aster size and spindle formation following washout. They use STLC treated cells presumably so that a mono-aster is measured. But in the figure, the CCDC66 siRNA cell shown has two spots of gamma tubulin - it did not form a monopolar spindle? Is this a common phenotype? is the quantification of microtubule length for single asters (presumably the centrosomes separate on washout)?5. The authors use knockdown and rescue to determine if the full-length protein, the microtubule binding C-terminal fragment and/or CCDC66 that is targeted to the centrosome with a PACT domain can rescue three aspects of the depletion phenotype: gamma tubulin at poles, spindle microtubule intensity and spindle length. The conclusion of the rescue experiments is that all three constructs can rescue these phenotypes, suggesting that CCDC66 at the centrosome is sufficient. In the discussion the authors state that "full length and C-terminal domain rescued these phenotypes to a greater extent than the CCDC66PACT" - although that is not clear from figure 7. Spindle length looks very similar in all cases and the PACT domain construct results more spindle tubulin. Are the differences in gamma level, spindle length and microtubule intensity for the full-length and PACT constructs significantly different (from each other) not just as compared to the depleted (and not rescued) cells? The three phenotypes measured all relate to the microtubule nucleating activity; are cytokinetic defects seen with the PACT containing construct? Is microtubule binding contributing to microtubule nucleation? I suppose resolving these issues will require use of constructs that contribute to a single function - nucleation or microtubule binding -- and are thus likely outside the scope of the present study. Nevertheless, the authors should be as clear as possible about the results of these experiments, which are impacted by possible overexpression of the rescue construct and additional centrosome binding in the case of the PACT construct. MinorI do not see CCD66 on astral microtubules in figure S1E; please provide single color image of the CCD66 fluorescence.The overexpression of CCD66 clearly results in artefacts as characterized in Figure 1D; thus, it is important that the level of expression be shown for rescue experiments and that the authors draw conclusions based on endogenous protein localizations.Page 9 the word "not" is missing when the interaction with mycBirA is described.In Movie 4 there are two mitotic cells; the one to the left looks like it undergoes apoptosis and the one on the left appears to undergo unequal division. In movie 5 there are also two mitotic cells; presumably the cell on the right is not depleted? Please add additional information.In figure 4 some panels have dots of two colors and others three; the colors represent different experimental trials—please clarify in the legend which experiments were repeated three (or two) times.Reviewer #2 (previous LSA reviewer): The revised version of the manuscript by Batman et al contains improvements that justify now consideration for a journal of higher visibility (such as PLOS Biology). In particular, the new data on endogenous CCDC66 represent a significant step forward. However, several of my earlier suggestions were apparently not followed: - I had criticized that spindle length in projected images appears shorter if spindles are tilted, and suggested that the length should be re-calculated from 3D data sets. Despite an appeasing response in which the authors promised that they will re-calculate , the data in the new Figure 4 are identical to the old version of the manuscript. - Rescue experiments for the cytokinesis phenotype were suggested. The authors state "As suggested by the reviewer, we have started performing experiments…. We will quantify … in the revised manuscript." However, the revised manuscript does not contain any such experiments. - I had criticized that in Fig. 5D, establishing a category of "prometaphase-like spindles" is not precise enough to describe the phenotype. Now, the authors labelled the same figure with the description "prometaphase spindles". This terminology is equally unsatisfactory, since spindle formation can be a long procedure even during a regular prometaphase, starting from monopolar arrays , transiting to somewhat "disorganized" spindles, and finishing with bipolar spindles. The authors must tell in descriptive terms what they see, for example "spindles with unfocused poles", or "spindles with low MT density", or other. The present classification makes little sense, since a typical "prometaphase spindle" does not exist. Overall, I am not satisfied with the revisions, and I suggest that additional improvements should be made. Reviewer #3 (previous LSA reviewer): Thank you to the authors to review the manuscript. They have addressed a critical number of experiments to reassure some of the reviewers' concerns;1-The authors have reassured the critical point of this work regarding the overexpression of CCD66. They show the endogenous level of CCDC66 by immunofluorescence and western blot in U2OS. Also, they demonstrate the localization to the endogenous levels at the spindle poles and microtubules. Moreover, they have examined the protein levels of endogenous versus exogenous in the U2OS stable cell line. 2- The authors have included some essential controls in the manuscript, replaced some representative images, and edited the manuscript to clarify some conclusions.The authors mention in the rebuttal letter the importance to clarify whether the microtubule bundling activity of the mNG-CCDC66 (570-948) is sufficient to rescue microtubule bundling associated mitotic phenotypes. I consider this phenotypic rescue experiment is essential, and I agree with the authors about the revision plan to discern this issue.As I mentioned in my previous revision, the results presented in this work are attractive to the general cell biological community, especially the centrosome and cilia communities. The authors are supported by solid evidence of their work, and their proposal strategy to address reviewers' comments is well conducted.Reviewer #4: In this manuscript the authors study the protein CCDC66 that was previously found to be localized on centrosome and MT and to be involved in mitotic spindle assembly (by the same team and others). Here the authors try to understand how CCDC66 participates to spindle organization. Although they found that CCDC66 is required to localize centrosome proteins important for spindle assembly (centrosome maturation) it remains puzzling to understand how it works. CCDC66 is also a MAP that bundles MT, and it is also as puzzling to reconcile these two functions. General commentsThe manuscript is very confusing, the experiments are well done but often one does not understand their choice and the order in which they are done.The results are often over-interpreted, sometimes even very badly interpreted.The manuscript shows interesting results but too preliminary as it is.CCDC66 seems to be involved in the nucleation of MTs from centrosomes. Without CCDC66 the bipolar spindle is formed, this spindle has a normal length but the global network of mitotic MTs is less dense. This is interesting but how does it work? There is no mechanism described that would explain this phenotype.Also, the controls are not shown in the right places. Two examples (1) siRNA depletion should appear to control for the specificity of the localization observed with the anti-CCDC66 antibody (2) rescue should demonstrate the specificity of the observed phenotype.However, the localization (fig1) is associated with a long discussion/interpretation on the possible function of CCDC66 while the control (siRNA) is only shown in figure S3.Same for the depletion (fig 4) which is very much discussed and interpreted while the control rescue is only shown fig 7.The manuscript must be rewritten in a more logical way and progression and additional experiment performed to propose a mechanism to explain the function of CCDC6. The manuscript also needs proofreading and correction of EnglishSpecific commentsPage 8 the authors say "Like other PCM proteins, CCDC66 formed resolvable rings at the PCM "I do not see any ring of CCDC66 on the spindle poles. May be in prometaphase but the resolution is not good enough to conclude. Page 9Is this sentence correct?CCDC66 co-pelleted with myc-BirA* fusions of CDK5RAP2, Cep192, Cep152 and gamma-tubulin, but with the negative control myc-BirA* .I think it should be but NOT with the negative control myc-BirAPage 9 again"FLAGminiTurbo did not co-pellet with myc-BirA* fusions of these positive interactions, andCCDC66 also did not co-pellet with the MT plus-end-tracking protein EB1, confirming thespecificity of its interactions with PCM proteins "One cannot draw such conclusion: not co-pelleting with EB1 is not enough to confirm the specificity of the interaction with the PCM proteins, it might only "suggest"Page 9 & 10"Taken together, our results suggest that CCDC66 functions during mitosis and cytokinesis by regulating centrosome maturation, MT nucleation and/or organization"This is an overinterpretation, I would rather write:"Taken together, the localization of CCDC66 during mitosis and cytokinesis led us to test potential functions of CCDC66 in the regulation of centrosome maturation, MT nucleation and/or organization"- ControlI would put the figure S3B in figure 1 as a control of the specificity of the antibodies used in fig1A (at least figure S3A and S3B in figure S1) this would reinforce the initial data.- Page 11 Microtubule densities after CCDC66 siRNA"In agreement, we noted that the MT arrays of the bipolar spindle, central spindle and midbody of CCDC66-depleted cells were disorganized and prominent defects in the assembly and organization of the bipolar spindle, central spindle and midbody such as disorganized MTs AND REDUCED MT INTENSITIES ."positioning. Relative to control cells, CCDC66"… depletion resulted in a minor decrease in average spindle lengths, which was measuredas pole-to-pole distance in metaphase cells ."I disagree, I don't see any differencePage 12"The tubulin fluorescence intensity at the spindle decreased about 0.6-fold in CCDC66-depleted cells relative to control cells ."This is pretty obvious and this is the phenotype the authors must concentrate on. - Page 12Fig 4B it appears that the whole MT network is less dense, MT nucleation or stability is affected. It seems that CCDC66 siRNA affect all MT not specifically Astral MT of K fibers."This result identifies CCDC66 as a regulator of K-fiber integrity, which explains the chromosome alignment defects and mitotic failure observed in CCDC66-depleted cells ."This looks again like an overinterpretation- Fig 5E, F and GThe images show a huge difference in the intensity of the centrosome labelling of gamma tub, pericentrin and CDK5RAP2 (IN PARTICULAR CDK5RAP2), whereas the data that represent mean ±SEM of two (Pericentrin) and three  independent experiments show very little difference. A comment might be necessary to say that the authors selected the best picture to illustrate the difference.- Fig S5"Notably, CCDC66-depleted cells had an increased number of MT "nucleating centers than control cells, suggesting possible activation of non-centrosomal MT nucleation pathways "This is a pretty good example demonstrating that numbers are useful, looking to the figure S5A one can find only one more nucleation point in cells depleted of CCDC66. I am not sure that it is a significant increase. However, the intensity of gamma tub labelling of each point increases in control cells but do not increase in CCDC66 depleted cells. This rather tells that nucleation of MT is affected. I would ask the authors to measure the intensity of gamma tub labelling of each point and compare with and without CCDC66.- Fig 6 and page 14"During the analysis of CCDC66-depleted cells stained for MTs, we noted that the cleavage furrow ingression is highly asymmetric and/or skewed in a significant number of cells ."highly asymmetric and/or skewed?The authors must be more preciseThe telophase cleavage furrow shown in Fig6C bottom is it asymmetric, skewed, bent …? What is the % of such images …Also it is difficult to draw any conclusion by only looking to and comparing the shape of the telophase cleavage furrow.I invite the authors to read the following paper :1625-9)- The rescueThese are very nice experiments, that strengthen the dataCCDC66-PACT rescue MT densities, this is surprising that the only localization at the centrosome is enough to rescue570-948 also works but the protein is all over the placeI would like to see WB showing the level of expression of the different proteins (NG CCDC66 1-948) (NG CCDC66-PACT) & (NG CCDC66 570-948) in the rescue experiments.« We performed rescue experiments for defective targeting of gamma-tubulin to thespindle poles, reduced spindle tubulin intensity and SHORTER SPINDLE LENGTH associated with CCDC66 loss »Well as I said in fig4A there is no detectable differences in the length of the spindle with or without CCDC66 26 Apr 2022AttachmentPointByPointResponse_final.pdfSubmitted filename: Click here for additional data file. 26 May 2022Dear Dr Firat-Karalar,Thank you for your patience while we considered your revised manuscript entitled "The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization" for publication as a Research Article at PLOS Biology. 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We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: -------------------------------------------------------------------------Reviewers' commentsRev. 1:This revised manuscript has been substantially improved. The points raised in the initial review have been addressed.Comments:1. for the assay of kinetochore fiber stability, the authors used a brief cold treatment to disassemble the non-kinetochore associated microtubules. Cells at metaphase have k-fiber microtubules that resist disassembly; cells prior to metaphase do not. Did the authors include MG132 to arrest cells at metaphase, thus avoiding the possibility that prometaphase cells are included in the analysis?2. what is the role of the N-terminal portion of CCDC66? The authors might state if there is a known role, as a reminder for the reader.3. Mispositioning and angle. The authors measure spindle tilt, a feature that they noticed was altered in the siRNA depleted cells. In the text they use "mispositioning" in several places to refer to this phenotype. Some cells position their spindle to one side of the cell, but to my knowledge the cells used in this study have centrally positioned spindles and the position of the spindle relative to the cell cortex was not actually measured. I think the term mis/positioning should not be used unless the authors mean that the spindle is off-center in a cell with a centrally located spindle. The feature measured was the angle relative to the substrate.4. Centrosome and spindle pole. I realize that these two terms are commonly used interchangeably, but they are not actually the same. The centrosome, consisting of the centrioles and PCM, localizes at the spindle pole for spindles in cells that have centrosomes.Spindle microtubules converge into the spindle pole and the components localized there are not centrosomal components. see: 10.1242/jcs.111.11.1477On the top of page 9, the authors say that CCDC66 is at the centrosome in interphase and at the spindle pole and microtubule based structures in mitosis. In this instance, "at the centrosome" might be more accurate.A bit later, they describe results using hi-resolution imaging to localize CCDC66 relative to markers of the "spindle pole" (top of page 10) and go on to say they used antibodies to PCM proteins. The first sentence in this section should read "centrosome" or mitotic centrosome, because that is what they are looking at. This section should be edited to refer to the centrosome.The presentation of the data and the writing have improved the manuscript.Rev. 2:The manuscript by Batman et al characterizes the role of the protein CCDC66 in mitosis. The authors made the novel discovery of CCDC66 acting in centrosomal recruitment of nucleation factors, and in the bundling of microtubules at the midbody during cytokinesis. The manuscript should be of interest to a large community of cell biologists.Compared to earlier versions of this submission, the authors made significant experimental improvements. All my earlier criticism has now been addressed and I have a favorable opinion on this manuscript.Rev. 3:I am happy that the authors have answered all of the points I raised during the first and second revisions.I consider that the authors have revised the manuscript addressing the major and minor concerns from the two revisions. They have added knowledge about the ciliar and nonciliar CCDC66 functions at the centrosomes and microtubules during cell division.Rev. 4:The authors have greatly improved their manuscript. This revised version has a much more logical flow and is now very enjoyable to read. The data have also been improved in a very significant way by adding a new set of experiments and data, with new antibodies for example and quantifications. Questionable data have been removed or my questions addressed by new experiments. The rescue experiments strengthen the data regarding the function of CCD66 in spindle assembly through nucleation and microtubule organization.This manuscript will certainly attract the attention of cell biologists working on microtubule spindle organization in mitosis.Excellent work! 8 Jun 2022AttachmentPointByPointResponse_SecondRound_final.pdfSubmitted filename: Click here for additional data file. 14 Jun 2022Dear Elif,On behalf of my colleagues and the Academic Editor, Renata Basto, I am pleased to say that we can accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. 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If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We are looking forward to publishing your study. Best wishes, RichardRichard Hodge, PhDAssociate Editor, PLOS Biologyrhodge@plos.orgPLOSEmpowering researchers to transform scienceCarlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdomplosbio.org I @PLOSBiology I BlogORCiD I California (U.S.) corporation #C2354500, based in San Francisco


Mortality in COVID-19 is mainly associated with respiratory failure, cytokine storm, and macrophage activation. Oxygenation and anti-inflammatory effects of Hyperbaric Oxygen Therapy (HBOT) suggest that it is a promising adjunct treatment for COVID-19. Repeated sessions of HBO with standard COVID-19 therapy were used to reduce the inflammation and increase oxygenation. We evaluated the safety and efficacy of HBOT in avoiding the replacement ventilation and/or ECMO and its effect on the inflammatory process. Twenty-eight moderate-to-severe COVID-19 patients were randomized into control or HBOT group. HBOT patients participated in 5 hyperbaric sessions (60 min). Before and after each session blood gas levels and vital parameters were monitored. Blood samples were collected for extended biochemical tests, blood morphology and immunological assays. There were 3 deaths in the control, no deaths in the HBOT group. No adverse events leading to discontinuation of HBOT were observed and patients receiving HBOT required lower oxygen delivery. We observed decrease in CRP, ferritin and LDH and increase in CD3 in HBOT group compared to control. This study confirmed the feasibility and safety of HBOT in patients with COVID-19 and indicated HBOT can lead to alleviation of inflammation and partial restoration of T cell responses. The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to a disease termed coronavirus disease-19 (COVID-19). The first cases of COVID-19 were identified in 2019 in China. The SARS-CoV-2 virus became known as highly infectious, spread between persons by droplet transmission. The disease rapidly spread throughout China, with its epicentre in Wuhan .Currently, there are lots of publications on the pathophysiology of COVID-19. From the clinical point of view, the pathomechanisms of the cellular effects of the virus resulting in cytokine release are important ,5. The mAs a viral infection, COVID-19 is characterized by type Th1 protective immune response , and memViral coagulopathy, which results in pulmonary embolism; venous, arterial, and capillary thrombosis; lung endothelial injury; and the accompanying pulmonary complications of pulmonary embolism, is the second form of the negative viral effects on humans. The exact mechanisms underlying this process are still unclear and require further investigation. Mortality in COVID-19 is mainly associated with acute, severe and progressive respiratory failure termed Acute Respiratory Distress Syndrome (ARDS). Early mortality rates for ARDS in COVID-19 patients in Wuhan Province, China, exceeded 90%. Most clinicians note the rapid progression of localised lung lesions leading to fibrosis. High levels of IL-6  and the Severe hypoxia leading to patient death is what all hypotheses have in common. In fact, reversal of hypoxia is an essential part of COVID-19 therapy ,16. It iHyperbaric Oxygen Therapy (HBOT) uses elevated environmental pressure (over 101.3 kPa = 1 ATA = 760 mmHg) to treat illness or injuries. In clinical practice, exposure pressure of 2.4–2.8 ATA lasting from 60 to 120 min is used, at which the arterial blood oxygen pressure is between 1800 mmHg and 2200 mmHg. During the patient’s exposure to hyperbaric conditions, 100% oxygen is usually used for breathing as it reaches all body tissues via the microcirculation, exerting therapeutic effects. Oxygen is a powerful trigger for cellular reactions . GeneralThe broad and physiological anti-inflammatory effects of HBO supported by extensive experimental and clinical evidence suggest that HBO is a promising treatment for COVID-19-associated ARDS ,26,27. OClinicalTrial.gov database (NCT04327505 and NCT04332081) and attempts to use this therapeutic modality in different countries as salvage therapy even in the absence of clinical trials (authors’ data).Until the launch of this study, there was no prospective randomised controlled trial (RCT) on the use of HBOT in COVID-19. Apart from a single published clinical case study , and reaOnly a few cases of HBOT use in severe cases of COVID-19 respiratory failure could be found in the literature ,40. All The research hypothesis of the project assumed that the use of repeated sessions of HBO as an adjunct therapy to standard COVID-19 therapy in moderate-to-severe patients can reduce the systemic and local inflammatory response in the lung tissue and pulmonary microcirculation and more rapidly extinguish the so-called cytokine storm ,42. PatiWe evaluated the safety and efficacy of HBOT in avoiding the need for replacement ventilation and/or ECMO and the rate of resolution of the inflammatory process as measured by inflammatory cell and inflammatory cytokine levels compared to a control group without HBOT.A randomized control trial was planned to be conducted as a prospective interventional study comparing standard treatment  with Hyperbaric Oxygen Therapy (HBOT) added as the adjunctive procedure to the standard treatment for COVID-19 (HBOT Group). The protocol was approved by the Ethical Committee, registered at EudraCT (2020-002722-90) and funded by the Polish Medical Research Agency (grant 2020/ABM/COVID19/0043). According to the power analysis we planned to recruit 60 patients over 1 year. After recruitment of 30 patients, who completed the screening phase and signed consent for participation in the clinical trial, the study was terminated due to inability to recruit more patients  and subjected to partial analysis. One patient was excluded due to instant improvement after steroid medication (ceased to meet the inclusion criteria). In the second case, exclusion criteria were identified before treatment onset. Finally, 28 patients completed the protocol. The protocol is presented on the g for 15 min at RT, then supernatants were aliquoted, frozen, and stored at −80 °C.For each blood collection, peripheral blood was drawn into a serum tube  and allowed to clot for 30 min at room temperature (RT). To obtain serum, blood samples were fractionated by centrifugation at 2000× g for 15 min at 4 °C, and 100 µL of sample was tested in duplicates on primary antibody coated and blocked 96 well plates. Detection was performed with the use of HRP substrate, TMB. Plate spectrophotometric reader  at 450 nm was used to measure optical density. Standard curve for recombinant IL-6 and blanked absorbance measurements were performed.Enzyme-linked immunosorbent assay  was used to measure IL-6 levels in serum samples. The assay was performed according to the manufacturer’s protocol. Briefly, serum samples were thawed on ice, centrifuged at 2500× ®, BD, Warsaw, Poland). Whole blood was used for extracellular markers immunofluorescence staining, 100 µL of blood was stained with anti-CD4-APC , anti-CD8-APC-AF700 , anti-CD3-APC-AF750  and anti-CD45-KrO  for 30 min at RT. Unstained blood was used as negative control for each patient, while single stainings were used for compensation settings and gating strategy. After staining, erythrocytes were lysed for 15 min at RT using BD FACSTM Lysing Solution  and washed twice in PBS. Data were analysed by flow cytometry using CYTOFLEX software v.2.3.0.84 . Debris was gated out on FCS/SSC dot plot and 30,000 events in “Cells” gate were acquired. Then, CD45+ cells were gated, followed by CD3+. Within the lymphocyte gate, the percentage of CD4+ and CD8+ cells was indicated.Peripheral blood samples were collected in EDTA-anticoagulated tube . The scheme of analysis included testing for within-group effects before and after the applied therapy—10 days after the launch of the study, as well as between-group comparisons of the first and last measurement. A temporal analysis from day 1 to day 5 of the study was performed only in the case of arterial blood gas levels. Data gaps in the final measurement were filled by the Last observation carried forward (LOCF) method. Due to lack of agreement with normal distribution in almost all results by group (Shapiro-Wilk test p < 0.05), non-parametric tests were used in the analyses. Intra-group analyses were performed using the Wilcoxon test, and inter-group analyses were conducted using the Mann-Whitney test. Bonferroni correction was applied in both tests. The analysis of arterial blood gas results for within-group (measurement) and between-group (HBOT vs. C group) effects was performed using the Friedman test.The analysis was performed using the R program (version 4.1.2.) with the rstatix library (Chip = 0.067). No adverse events (AEs) leading to discontinuation of any single HBOT sessions were observed in the HBOT group . Twenty-seven patients had comorbidities. The most common diagnoses were hypertension and insulin-dependent diabetes mellitus. There was no statistically significant difference in the prevalence of comorbidities between the C and the HBOT group . In 5 patients , COVID-19 viral pneumonia was the only diagnosis. Most patients  had several comorbidities (including one excluded). No statistically significant difference was observed between groups C and HBOT in age , or sex . Patient status according to the National Early Warning Score (NEWS) was similar in both groups at baseline  and final HBOT measurement: M = 1.72, SD = 0.99; C: M = 2.36, SD = 1.8; p = 0.962). As part of their treatment, all patients received subcutaneous anticoagulants and corticosteroids. Twenty-seven patients (including 2 patients excluded from the study) received antibiotic therapy, 5 patients were treated with remdesivir  and one patient was treated with tocilizumab (in the HBOT group).Thirty patients of the Military Medical Institute, including 24 males and 6 females, aged 24–78 years (mean age 55 ± 13.4), hospitalised for SARS-CoV-2 infection from 1 March 2021 to 3 February 2022, participated in the study. All subjects signed a consent to participate in the study. Two patients were excluded due to the failure to meet the inclusion criteria. In the control group, 1 patient refused to continue participation on day 10, and 1 patient, a woman aged 51 years, refused to participate in the study on day 1. Following randomisation, 14 patients were assigned to the HBOT group, and 14 patients were placed in the Control group (C). In most cases, the HBOT started within 24 h from randomization, which was on average 5 days after the initial onset of symptoms. There were 3 deaths in the control (C) group, and no deaths in the HBOT group. The difference in death rate did not reach statistical significance , PiO2/FiO2: F = 6.4; p = 0.171). Temporal analysis within groups also showed no significant differences in SpO2 , while for the Horowitz coefficient in the HBOT group, the temporal effect was at trend level (p = 0.068), in group C it was not significant (p = 0.835). The change in parameters over time is shown in The degree of lung injury was determined, among other things, using the Horowitz factor , and the severity of normobaric oxygen therapy was measured by determining the amount of oxygen used. One of the therapeutic goals was to maintain normal oxygenation as measured by blood saturation (SpO2) levels. In fact, this was achieved in all patients. Statistical analysis showed that mean SpO2 and PiO2/FiO2 values did not differ significantly between groups throughout the study . An analp < 0.00001). In contrast, the intragroup differences in oxygen delivery by measurement were not statistically significant (day 1–5 before or after HBOT session), but on the second day after the session  and on day 5 before the session  the values were at the border of significance or at the trend level.Oxygen supply was tailored to individual patient needs to avoid hypoxemic events with SpO2 < 92%. In 6 patients, no passive oxygen therapy was used at different stages of the study, 13 patients used an oxygen reservoir mask during all or part of the measurements, while 2 patients breathed through a simple mask, one patient was administered oxygen via a mask with an oxygen reservoir and high-flow intranasal oxygen therapy (HFNOT) and one patient used a mask and oxygen sniffers, 6 patients used intranasal cannulas, and 1 patient required the use of a ventilator . Comparing the mean oxygen delivery throughout the study, a significantly lower oxygen delivery can be observed during treatment in the HBOT group compared to the control group . The anap = 0.042), ferritin [mg/mL]: W = 28, p = 0.045) and LDH [µ/L]: W = 28, p = 0.045 were observed vs. baseline in the HBOT group, but no difference was observed for D-Dimers [mg/mL]: p = 0.27) and procalcitonin (PCT [mg/mL]): p = 0.108) , D-Dimers [mg/mL]: p > 0.999; PCT [mg/mL]: p = 0.544).No statistically significant differences in biochemical tests  were observed between the HBOT and C groups at baseline and follow-up . At the = 0.108) . In group > 0.05)—p = 0.045), lymphocytes  and statistically significant decreases in neutrophils  were observed in both groups (p > 0.05). Other parameters  remained unchanged in both groups (p > 0.05).No statistically significant CBC differences  were observed between HBOT and C groups at baseline (h groups . These cp > 0.05)—p = 0.042). Other parameters  remained unchanged in both groups (p > 0.05) , CD4 or CD8 lymphocyte percentages between HBOT and C groups at baseline ( > 0.05) .p = 0.044, but not after study completion (IL-6 (pg/mL): U = 118; p = 0.74)—p > 0.999; C: p = 0.161) (p = 0.298) and in the final measurement (p > 0.999).Statistically significantly higher levels of interleukin 6 (IL-6) were observed in C group vs. HBOT group before the study (IL-6 (pg/mL): U = 142; = 0.161) . After ep = 0.067) to such significance. With a small sample size, this strongly suggests beneficial effects of HBO on reduction of mortality.This study confirmed the feasibility and safety of HBOT in patients with moderate-to-severe SARS-CoV-2 pneumonia during the period of high infectiousness and requiring continuous delivery of high oxygen flows. No cases of life-threatening complications were observed in the HBOT group. After treatment of half of the planned population, persistence of significant mortality was observed only in the non-HBO group , whereas no mortality was reported in the HBO group  despite the same disease severity parameters in both groups at randomisation. This difference did not reach statistical significance but was close , but no significance for the difference in mortality (p = 0.14). As in other similar work, the final conclusion was “Though limited by its study design, our results demonstrate the safety of hyperbaric oxygen among COVID-19 patients and strongly suggests the need for a well-designed, multi-centre randomized control trial.”The first RCT reporting the use of HBO as adjunctive treatment in patients with COVID-19 was published on 14 December 2021 by Cannellotto et al. . For thiIn our study, we decided to focus on the biochemical, cellular and cytokine effects of HBOT in COVID-19 patients. At the time of preparation of the study protocol, there was no published data on immunological aspects of HBOT in COVI-19. While we are still awaiting some other studies to be reported, the only other research on HBOT efficacy as an adjuvant for the Systematic Inflammation Reduction in patients with SARS-CoV-2 infection was published by the Mexican group . They coNo statistically significant differences were observed between groups C and HBOT in the baseline values of the parameters tested , demonstrating the effectiveness of randomisation.When concerning IL-6, a statistically significant difference in Interleukin 6 (IL-6) levels was observed between groups C and HBOT at baseline, while no statistically significant change was observed in either group. IL-6 levels were 2-fold higher in the control group. High levels of IL-6 were observed in patients who died . After performing an additional analysis that did not include deceased patients, no statistically significant differences were found between groups or time points.In both C and HBOT groups, the pre-set oxygenation parameters based on SpO2 and PaO2/FiO2 were maintained, which proves treatment efficacy. In the HBOT group, this was achieved by using lower passive oxygen therapy flows. However, the statistical difference in the amount of oxygen used between groups C and HBOT does not translate into therapeutic efficacy. In fact, it is well known fact for all clinician that using SpO2 or PaO2/FiO2 index in non-intubated patients is prone to even slight fluctuations in oxygen delivery, due to patient movement, speaking or drinking.A statistically significant increase in the percentage of eosinophilia and lymphocytes and a decrease in neutrophils were observed in both groups during the study. These changes did not differ between C and HBOT groups.During the study, statistically significant changes only in the HBOT group included decreases in CRP, ferritin and LDH and increases in CD3. This confirms that indeed HBOT influences at least some biochemical and cellular parameters of COVID-19 patients.The most interesting, and not yet reported fact is a change in CD3+ percentage observed only in HBOT group, and not in C group. Lower CD3+ percentage has been shown as a predictor of COVID-19 severity . In our Additionally, though limited by its study design, our results demonstrate the safety of hyperbaric oxygen among COVID-19 patients and strongly suggests the need for a well-designed, multi-centre randomized controlled trial.A relatively up-to-date inventory of registered scientific studies has been published by UHMS (UHM 2020). Also published are the positions of the major scientific organisations involved in hyperbaric medicine on the use of HBOT in patients with COVID-19: EUBS/ECHM  and UHMSThe next scientific question could be related with the possible use of HBOT in so-called “long COVID-19” . We are The present study also demonstrates the feasibility of successful and safe HBO sessions in patients with moderate-to-severe SARS-CoV-2 pneumonia during a period of high infectiousness and requiring continuous supply of high oxygen flows. The issue of safety of the staff caring for patients on high oxygen flows or in the confined space of a hyperbaric chamber has also been raised in other publications ,66,67,68We acknowledge the limitations of our study. One is that the small sample size restricts the study results. Still, it answers the primary endpoint on HBOT safety in COVID-19 patients and on the secondary endpoints related to affecting the immunological status of COVID-19 patients by the hyperbaric oxygenation. However, it is not powered enough to either confirm or deny the clinical efficacy of HBOT in the general population of patients with COVID-19. Another limitation is that the treating physicians determined standard care for COVID-19 in both groups; thus, it is possible that differences in standard care throughout the trial enrolment period could influence outcomes.This analysis of our prospective RCT on using HBOT in severe COVID-19 confirmed the effectiveness of randomisation (except for IL-6), showed a clear trend towards fewer deaths in the HBOT group, confirmed the possibility of safely conducting HBOT in this patient group, showed a lower need for normobaric oxygenation in the group of patients treated with HBOT and demonstrated significant beneficial effects of HBOT on CRP, ferritin, LDH and CD3.Considering the obtained results, including the positive response to the question regarding the effect of HBOT on the inflammatory response, the declining rate of patient recruitment, the changing phenotype of the SARS-CoV-2 virus and the changing immune status of the patients due to COVID-19 or subsequent doses of vaccination, it was concluded that continuing the study according to the previous protocol has little chance of increasing the strength of validity of the results achieved. It was therefore decided that this study should be terminated.